The present invention concerns pharmaceutical compounds and compositions that are useful as tissue protectants, such as neuroprotectants and cardioprotectants. The compounds and compositions may be used, for example, in the treatment of acute ischemic neurological insults or chronic neurodegenerative diseases.
Permanent injury to the central nervous system (CNS) occurs in a variety of medical conditions, and has been the subject of intense scientific scrutiny in recent years. It is known that the brain has high metabolic requirements, and that it can suffer permanent neurologic damage if deprived of sufficient oxygen (hypoxia) for even a few minutes. In the absence of oxygen (anoxia), mitochondrial production of ATP cannot meet the metabolic requirements of the brain, and tissue damage occurs. This process is exacerbated by neuronal release of the neurotransmitter glutamate, which stimulates NMDA (N-methyl-D-aspartate), AMPA (xcex1-amino-3-hydroxy-5-methyl-4-isoxazole propionate) and kainate receptors. Activation of these receptors initiates calcium influx into the neurons, and production of reactive oxygen species, which are potent toxins that damage important cellular structures such as membranes, DNA and enzymes.
The brain has many redundant blood supplies, which means that its tissue is seldom completely deprived of oxygen, even during acute ischemic events caused by thromboembolic events or trauma. A combination of the injury of hypoxia with the added insult of glutamate toxicity is therefore believed to be ultimately responsible for cellular death. Hence if the additive insult of glutamate toxicity can be alleviated, neurological damage could also be lessened. Anti-oxidants and anti-inflammatory agents have been proposed to reduce damage, but they often have poor access to structures such as the brain (which are protected by the blood brain barrier).
Given the importance of the NMDA, AMPA and kainate receptors in the mechanism of injury, research efforts have focused on using antagonists to these receptors to interfere with the receptor mediated calcium influx that ultimately leads to cellular death and tissue necrosis. In vitro studies using cultured neurons have demonstrated that glutamate receptor antagonists reduce neurotoxicity, but NMDA and AMPA/kainate receptor antagonists have different effects. Antagonists to NMDAr prevent neurotoxicity if present during the glutamate exposure period, but are less effective if added after glutamate is removed. In contrast, AMPA/kainate receptor antagonists are not as effective as NMDA antagonists during the glutamate exposure period, but are more effective following glutamate exposure.
Some of the research on these antagonists has focused on cannabinoids, a subset of which have been found to be NMDA receptor antagonists. U.S. Pat. No. 5,538,993 (3S,4S-delta-6-tetrahydrocannabinol-7-oic acids), U.S. Pat. No. 5,521,215 (sterospecific (+) THC enantiomers), and U.S. Pat. No. 5,284,867 (dimethylheptyl benzopyrans) have reported that these cannabinoids are effective NMDA receptor blockers. U.S. Pat. No. 5,434,295 discloses that the 1,1 dimethylheptyl (DMH) homolog of [3R,4R]-7-hydroxy-xcex946THC (known as HU-210) is a superpotent cannabinoid receptor agonist with cannabinomimetic activity two orders of magnitude greater than the natural xcex949 THC. The HU-210 dimethylheptyl cannabinoid, has severe side effects, including fatigue, thirst, headache, and hypotension. J. Pharmacol. Sci. 60:1433-1457 (1971). Subjects who received this synthetic cannabinoid with a dimethylheptyl group experienced marked psychomotor retardation, and were unwilling or incapable of assuming an erect position.
In contrast to HU-210, the (xe2x88x92)(3R,4R) THC-DMH enantiomer (known as HU-211) displays low affinity to the cannabinoid receptors, but retains NMDA receptor antagonist neuroprotective activity. 
THC (tetrahydrocannabinol) is another of the cannabinoids that has been shown to be neuroprotective in cell cultures, but this protection was believed to be mediated by interaction at the cannabinoid receptor, and so would be accompanied by undesired psychotropic side effects. 
Although it has been unclear whether cannabimimetic activity plays a role in neuroprotection against glutamate induced neurological injury, the teaching in this field has clearly been that a cannabinoid must at least be an antagonist at the NMDA receptor to have neuroprotective effect. Hence cannabidiol (2-[3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol or CBD), a cannabinoid devoid of psychoactive effect (Pharm. Rev. 38:21-43, 1986), has not been considered useful as a neuroprotectant. Cannabidiol has been studied as an antiepileptic (Carlini et al., J. Clin. Pharmacol. 21:417S-427S, 1981; Karler et al., J. Clin. Pharmacol. 21:437S-448S, 1981, Consroe et al., J. Clin Phannacol. 21:428S-436S, 1981), and has been found to lower intraocular pressure (Colasanti et al, Exp. Eye Res. 39:251-259, 1984 and Gen. Pharmac. 15:479-484, 1984). 
No signs of toxicity or serious side effects have been observed following chronic administration of cannabidiol to healthy volunteers (Cunha et al., Pharmacology 21:175-185, 1980), even in large acute doses of 700 mg/day (Consroe et al., Pharmacol. Biochem. Behav. 40:701-708, 1991) but cannabidiol is inactive at the NMDA receptor. Hence in spite of its potential use in treating glaucoma and seizures, cannabidiol has not been considered a neuroprotective agent that could be used to prevent glutamate induced damage in the central nervous system.
It is an object of this invention to provide a new class of antioxidant drugs, that have particular application as neuroprotectants, although they are generally useful in the treatment of many oxidation associated diseases.
Yet another object of the invention is to provide a subset of such drugs that can be substantially free of psychoactive or psychotoxic effects, are substantially non-toxic even at very high doses, and have good tissue penetration, for example crossing the blood brain barrier.
It has surprisingly been found that cannabidiol and other cannabinoids can function as neuroprotectants, even though they lack NMDA receptor antagonist activity. This discovery was made possible because of the inventor""s recognition of a previously unanticipated antioxidant property of the cannabinoids in general (and cannabidiol in particular) that functions completely independently of antagonism at the NMDA, AMPA and kainate receptors. Hence the present invention includes methods of preventing or treating diseases caused by oxidative stress, such as neuronal hypoxia, by administering a prophylactic or therapeutically effective amount of a cannabinoid to a subject who has a disease caused by oxidative stress.
The cannabinoid may be a cannabinoid other than THC, HU-210, or other potent cannabinoid receptor agonists. The cannabinoid may also be other than HU-211 or any other NMDA receptor antagonist that has previously been reported. A potent cannabinoid receptor agonist is one that has an EC50 at the cannabinoid receptor of 50 nM or less, but in more particular embodiments 190 nM or 250 nM or less. In disclosed embodiments the cannabinoid is not psychoactive, and is not psychotoxic even at high doses. In some particularly disclosed embodiments, the cannabinoid is selected from the group: 
where A is aryl, and particularly 
but not a pinene such as: 
and the R1-R5 groups are each independently selected from the groups of hydrogen, lower substituted or unsubstituted alkyl, substituted or unsubstituted carboxyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alcohol, and substituted or unsubstituted ethers, and R6-R7 are H or methyl. In particular embodiments, there are no nitrogens in the rings, and/or no amino substitutions on the rings.
In other embodiments, the cannabinoid is one of the following: 
where there can be 0 to 3 double bonds on the A ring, as indicated by the optional double bonds indicated by dashed lines on the A ring. The C ring is aromatic, and the B ring can be a pyran. Particular embodiments are dibenzo pyrans and cyclohexenyl benzenediols. Particular embodiments of the cannabinoids of the present invention may also be highly lipid soluble, and in particular embodiments can be dissolved in an aqueous solution only sparingly (for example 10 mg/ml or less). The octanol/water partition ratio at neutral pH in useful embodiments is 5000 or greater, for example 6000 or greater. This high lipid solubility enhances penetration of the drug into the CNS, as reflected by its volume of distribution (Vd) of 1.5 L/kg or more, for example 3.5 L/kg, 7 L/kg, or ideally 10 L/kg or more, for example at least 20 L/kg. Particular embodiments may also be highly water soluble derivatives that are able to penetrate the CNS, for example carboxyl derivatives.
R7-18 are independently selected from the group of H, substituted or unsubstituted alkyl, especially lower alkyl, for example unsubstituted C1-C3 alkyl, hydroxyl, alkoxy, especially lower alkoxy such as methoxy or ethoxy, substituted or unsubstituted alcohol, and unsubstituted or substituted carboxyl, for example COOH or COCH3. In other embodiments R7-18 can also be substituted or unsubstituted amino, and halogen.
The cannabinoid has substantially no binding to the NMDAr (for example an IC50 greater than or equal to 5 xcexcM or 10 xcexcM), has substantially no psychoactive activity mediated by the cannabinoid receptor (for example an IC50 at the cannabinoid receptor of greater than or equal to 300 nM, for example greater than 1 xcexcM and a Ki greater than 250 nM, especially 500-1000 nM, for example greater than 1000 nM), and antioxidant activity, as demonstratable by the Fenton reaction or cyclic voltametry.
In other particular embodiments, the cannabinoids are one of the following: 
where R19 is substituted or unsubstituted alkyl, such as lower alkyl (for example methyl), lower alcohol (such as methyl alcohol) or carboxyl (such as carboxylic acid) and oxygen (as in xe2x95x90O); R20 is hydrogen or hydroxy; R21 is hydrogen, hydroxy, or methoxy; R22 is hydrogen or hydroxy; R23 is hydrogen or hydroxy; R24 is hydrogen or hydroxy; R25 is hydrogen or hydroxy; and R26 is substituted or unsubstituted alkyl (for example n-methyl alkyl), substituted or unsubstituted alcohol, or substituted or unsubstituted carboxy.
In yet other embodiments of the invention, the cannabinoids are 
wherein numbering conventions for each of the ring positions are shown, and R27, R28 and R29 are independently selected from the group consisting of H, unsubstituted lower alkyl such as CH3, and carboxyl such as COCH3. Particular examples of nonpsychoactive cannabinoids that fall within this definition are cannabidiol and 
and other structural analogs of cannabidiol.
In more particular embodiments, the cannabinoid is used to prevent or treat an ischemic or neurodegenerative disease in the central nervous system of a subject, by administering to the subject a therapeutically effective amount of a cannabinoid to protect against oxidative injury to the central nervous system. The cannabinoid may be any of the compounds set forth above, or more specifically 
wherein R27, R28 and R29 are independently selected from the group consisting of H, lower alkyl such as CH3, and carboxyl such as COCH3, and particularly wherein
a) R27=R28=R29=H
b) R27=R29=H; R28=CH3 
c) R27=R28=CH3; R29=H
d) R27=R28=COCH3; R29=H
e) R27=H; R28=R29=COCH3 
When R27=R28=R29=H, then the compound is cannabidiol. When R27=R29=H and R28=CH3, the compound is CBD monomethyl ether. When R27=R28=CH3 and R29=H, the compound is CBD dimethyl ether. When R27=R28=COCH3 and R29=H, the compound is CBD diacetate. When R27=H and R28=R29=COCH3, the compound is CBD monoacetate. The ischemic or neurodegenerative disease may be, for example, an ischemic infarct, Alzheimer""s disease, Parkinson""s disease, Down""s syndrome, human immunodeficiency virus (HIV) dementia, myocardial infarction, or treatment and prevention of intraoperative or perioperative hypoxic insults that can leave persistent neurological deficits following open heart surgery requiring heart/lung bypass machines, such as coronary artery bypass grafts (CABG).
The invention also includes an assay for selecting a cannabinoid to use in treating a neurological disease by determining whether the cannabinoid is an antioxidant. Once it has been determined that the cannabinoid is an antioxidant, an antioxidant effective amount of the cannabinoid is administered to treat the neurological disease, such as a vascular ischemic event in the central nervous system, for example the type caused by a neurovascular thromboembolism. Similarly, the method of the present invention includes determining whether a disease is caused by oxidative stress, and if the disease is caused by oxidative stress, administering the cannabinoid in a therapeutically effective antioxidant amount.
The invention also includes identifying and administering antioxidant and neuroprotective compounds (such as cannabidiol) which selectively inhibit the enzyme activity of both 5- and 15-lipoxygenase more than the enzyme activity of 12-lipoxygenase. In addition, such compounds posses low NMDA antagonist activity and low cannabinoid receptor activity. Assays for selecting compounds with the desired effect on lipoxygenase enzymes, and methods for using identified compounds to treat neurological or ischemic diseases are also provided. Such diseases may include a vascular ischemic event in the central nervous system, for example a thromboembolism in the brain, or a vascular ischemic event in the myocardium. Useful administration of the compounds involves administration both during and after an ischemic injury.
These and other objects of the invention will be understood more clearly by reference to the following detailed description and drawings.